Covalent modification by isoprenoid lipids (prenylation) is a critical post-translational event for many proteins involved in cellular signaling and cancer. The primary goal of this research program is to design and test prenyl function inhibitors and to identify and characterize proteins that are farnesylated in vivo. The studies outlined in this proposal will result in the preparation of new tools to probe the specificity of the prenyltransferases protein- farnesyltransferase (FTase) and protein-geranylgeranyltransferase (GGTase-I), enable further development of prenyl function inhibitors and identify new prenylated proteins as potential targets for therapeutic intervention. By varying the chemical structure of the prenyl lipid, we are developing reagents to probe the biological function of the posttranslational modification. Taking advantage of the fact that the prenyl group forms a substantial part of the peptide substrate binding site in the prenyltransferases has allowed us to develop peptide selective inhibitors of prenyl function. These unnatural analogues may allow for the selective interference with specific prenylation targets and may provide lead compounds to alleviate the potential toxicity associated with complete inhibition of protein prenylation. In particular, the unnatural analogues may be useful to obtain a more complete understanding of the role that alternative prenylation plays in Farnesyl transferase inhibitor (FTI) evasion by oncogenes such as K-Ras. Critical to understanding the clinical effects of existing and future FTIs and geranylgeranyl transferase inhibitors (GGTIs), is the identification of in vivo substrates of FTase and GGTase-I. Our innovative strategy of using unnatural, transferable prenyl analogues and analogue specific monoclonal antibodies to identify prenylated cellular proteins will provide valuable information on the cellular targets of inhibitors of prenylation. We expect these studies to result in the identification of previously unknown prenylated proteins. The specific aims of this project are: 1) Synthesis of farnesyl and geranylgeranyl diphosphate analogues to study protein prenylation. 2) Screening these compounds for ubstrate specificity of FTase and GGTase-I and development of prenyl function inhibitors. 3) Identification of prenylated proteins in cells. The results from these studies may provide leads to new molecules to treat cancer and also identify new molecular targets to develop anti-cancer therapies. Substantial evidence points to the central role of proteins normally modified by a prenyl group in cancer progression. We propose to design and test prenyl function inhibitors and to identify and characterize proteins that are modified with prenyl groups. The results from these studies may provide leads to new molecules to treat cancer and also identify new molecular targets to develop future anti-cancer therapies.